Microsatellite analysis of variation among wild, domesticated, and genetically improved populations of blunt snout bream (Megalobrama amblycephala).

In the present study, the genetic diversity of one selected strain (Pujiang No. 1), two domesticated populations (GA and HX) and four wild populations (LZ, YN, SS and JL) of blunt snout bream (Megalobrama amblycephala) was analyzed using 17 microsatellite markers. The results showed that an average of 4.88-7.65 number of alleles (A); an average of 3.20-5.33 effective alleles (Ne); average observed heterozygosity (Ho) of 0.6985-0.9044; average expected heterozygosity (He) of 0.6501-0.7805; and the average polymorphism information content (PIC) at 0.5706-0.7226. Pairwise FST value between populations ranged from 0.0307-0.1451, and Nei's standard genetic distance between populations was 0.0938-0.4524. The expected heterozygosities in the domesticated populations (GA and HX) were significantly lower than those found in three wild populations (LZ, SS and JL), but no difference was detected when compared with the wild YN population. Likewise, no difference was found between the four wild populations or two domesticated populations. The expected heterozygosity in Pujiang No. 1 was higher than the two domesticated populations and lower than the four wild populations. Regarding pairwise FST value between populations, permutation test P-values were significant between the GA, HX and PJ populations, but not between the four wild populations. These results showed that the expected heterozygosity in the selected strain of blunt snout bream, after seven generations of selective breeding, was lower than that of wild populations, but this strain retains higher levels of genetic diversity than domesticated populations. The genetic differences and differentiation amongst wild populations, domesticated populations and the genetically improved strain of blunt snout bream will provide important conservation criteria and guide the utilization of germplasm resources.

Complete mitochondrial genome of blackchin tilapia Sarotherodon melanotheron (Perciformes, Cichlidae).

Blackchin tilapia, Sarotherodon melanotheron, is a highly salt-tolerant species in tilapias. In this paper, the complete mitochondrial genome of S. melanotheron was determined first. The mitogenome (16,627 bp) had the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding, 22 tRNA, 2 rRNA genes, and 1 putative control region. It shared 95.1%, 93.2%, and 92.2% mitogenome sequence with Oreochromis aureus, Oreochromis niloticus, and Oreochromis mossambicus, respectively.

Possible genetic reproductive isolation between two tilapiine genera and species: Oreochromis niloticus and Sarotherodon melanotheron.

Successful crossbreeding between Oreochromis niloticus and Sarotherodon melanotheron to produce a commercial hybrid has been difficult. The karyotypes and isoenzyme of these two species and their reciprocal hybrids (O. niloticus female × S. melanotheron male, S. melanotheron female × O. niloticus male, the last not included in the isoenzyme study) were investigated via metaphase chromosomes obtained from head kidney cells and electropherogram of lactate dehydrogenase (LDH) isoenzymes from the liver, kidney, white muscle, heart, and eye balls. The diploid chromosome number (2n=44) and the fundamental number (NF=50) of the four tilapia genotypes were the same. However, the karyotype of O. niloticus had three pairs of sub-metacentric (sm), twelve pairs of sub-telocentric (st), and seven pairs of telocentric (t) chromosomes, while S. melanotheron had one pair of metacentric (m), two pairs of sm, 12 pairs of st, and seven pairs of t chromosomes. The reciprocal hybrids both showed a mixed karyotype range between their parents: 0.5 pair of m, 2.5 pairs of sm, 12 pairs of st, and seven pairs of t chromosomes. In view of the electropherogram of isozymes, only the LDH of the kidney showed significant clear bands, with five bands in O. niloticus, three bands in S. melanotheron, and duplicated six bands in the hybrids. The bands varied depending on their activities and mobilities. We considered that the differences in karyotype and isoenzyme were related to the genetic mechanism for post-mating isolation, and provided some additional basic genetic background of their taxonomy.

[cDNA cloning and tissue-differential expression of Na+/K+/2Cl(-)-cotransporter 1-alpha isoform in Sarotherodon melanotheron].

The gills are the major apparatus for osmoregulation in fish to acclimate the changes of salinities. Na+/K+/2Cl(-)cotransporter 1-alpha (NKCC1 alpha) is one of the key ion cotransporter locoalized in gill chloride cells which has been associated with the maintence of osmotic homeostasis. The transport process mediated by NKCC1 alpha is characterized by electroneutrality with a stoichiometry of 1Na:1K:2Cl. Sarotherodon melanotheron is one of the most euryhaline teleosts able to withstand variations in environmental salinity ranging from freshwater to hyper-saline waters. In this study, the reverse transcription-polymerase chain reaction and rapid amplification of 3' and 5'cDNA ends methods were used to identify the full cDNA of the NKCC1 alpha with an Open Reading Frame which contains 1 151aa of S.melanotheron. The amino acid multiple alignment and phylogenetic analysis showed that this isoform is more similar with isoforms in Oreochromis mossambicus, Salmo salar and Anguilla anguilla, and there is the highest homologous of 99% between Sarotherodon and Mossambique. The predicted protein secondly structure of NKCC1 alpha contains 10 transmenbrane domains, which were highly conserved in sequences and locoalization sites relatively to other species. The quantitative real time polymerase chain reaction (qRT-PCR) assay was developed to estimate the mRNA expression levels in gill, liver, intestine and kidney in freshwater, the results showed a tissue-specific model. Furthermore, the sanility significantly affects the relative expression level of NKCC1 alpha mRNA in gill with a 4.9 times higher in 136 salinity water than that in 0 salinity. The results suggest that the NKCC1 alpha is closely related to the salt tolerance in S.melanotheron.

Morphological changes of silver and bighead carp in the Yangtze River over the past 50 years.

Multivariate analysis was adopted to analyze 30 morphometrical characteristics of 121 one-year-old juvenile silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis) bred during the 1950s ("the former population") and 2008 ("the current population") and collected from the middle reach of the Yangtze River. The average discriminant accuracies of the former and current silver and bighead carp population were 94.2% and 98.0%, respectively. Discriminant analysis also revealed that significant differences in morphology occurred between the former and current populations of both carp in overall characteristics. One-way analysis of variance indicated that between former and current populations, silver carp showed highly significant differences (P<0.01) in twelve of their characteristics and significant differences (P<0.05) in eight of their characteristics, while bighead carp showed highly significant differences (P<0.01) in eight of their characteristics and significant differences (P<0.05) in eight of their characteristics. Six head morphology variables of the current silver and bighead carp were significantly or highly significantly larger than the former populations; fourteen characteristics of silver carp and ten characteristics of bighead carp of the current populations, mainly reflecting truck and tail morphology, were significantly or very significantly smaller than the former populations. Our results indicate that silver and bighead carp have developed a larger head and smaller truck and tail during the last 50 years. Due to such morphological changes, it seems apparent that the heads of these fish species need to be considered in regards to human diets, particularly in relation to economic and nutritious value.

RAPD-SCAR Markers for Genetically Improved NEW GIFT Nile Tilapia (Oreochromis niloticus niloticus L.) and Their Application in Strain Identification.

The NEW GIFT Nile tilapia (Oreochromis niloticus niloticus L.) is a nationally certificated new strain selected over 14 years and 9 generations from the base strain of GIFT Nile tilapia, introduced in 1994. This new variety has been extended in most of areas of China. The management of genetically improved strains, including the genetic markers for identification is needed urgently. RAPD analysis was conducted and their conversion to SCAR markers was developed. From NEW GIFT Nile tilapia, two strain-specific RAPD bands, S(304 )(624 bp ) and S(36 )(568 bp ) were identified. The strain-specific RAPD bands were gel-purified, cloned, and sequenced. Locus-specific primers were then designed to amplify the strain-specific bands. PCR amplification was conducted to test the variations in allele frequencies of two converted SCAR markers among the NEW GIFT Nile tilapia and its base strains, as well as 7 additional farmed strains worldwide. The frequency of SCAR marker I (553 bp) was 85.7% in NEW GIFT Nile tilapia, but 16.7% in the base strain. The frequency of SCAR marker II (558 bp) was 91.4% in NEW GIFT Nile tilapia, but 0% - 70% in the 7 other strains. In order to confirm the utility of these two markers, an examination was conducted for a wild population from Egypt, resulted the frequency of SCAR I and II was 10% and 70%, respectively, much lower than that of New GIFT strain. The increase in allele frequency of these two SCAR markers suggests that these markers might be genetically linked to the quantitative trait loci (QTL) underlining the performance traits by long term selection, and indicate the bright potential of SCAR marker technology for tracking generations during selection progress and for distinguishing among genetically improved strain and other strains.

Ontogenetic changes in meristic measurements of silver carp and bighead carp.

The study analyzed 30 exterior meristic characters for 255 silver and bighead carp samples of 1- to 5-year-old collected from the National Primary Breeding Farm at Laojianghe Lake at the Middle Reach of the Yangtze River. Multivariate analysis was performed. In silver carp, the Euclidean distance was the greatest between the 1-year-old group and other age groups. Silver carp individuals were correctly classified at 98.0% accuracy with a discriminant function established by discriminant analysis based on meristic measurements. Similarly, bighead carp had the greatest distance between 1- to 2-year-old group and other age groups. Individuals of bighead carp were correctly classified at 90.7% accuracy by the discrimination function. The data showed that morphological transformation occurred during the life history of silver and bighead carp development. Eighteen meristic measurements showed highly significant differences, while four showed a significant difference between the two silver carp groups. Ten parameters decreased, while twelve measurements increased during development. In bighead carp, fourteen parameters were significantly different, while three parameters were significantly different between the two groups. Twelve parameters were significantly decreased and another five were increased during development. The results suggest allometric growth should be taken into account when identifying species, analyzing population differences and establishing germplasm standards based on morphology.

Development of microsatellite markers for blunt snout bream Megalobrama amblycephala using 5'-anchored PCR.

A rapid method for isolating microsatellite loci in blunt snout bream, based on the 5'-anchored polymerase chain reaction technique, revealed 522 microsatellite loci (consisting of 442 dinucleotide, 4 trinucleotide and 76 tetranucleotide repeats). Of the 25 loci characterized, 10 turned out to be highly polymorphic. The number of alleles per locus ranged from 3 to 17 while the expected heterozygosity ranged from 0.4899 to 0.9355 in population of selected strain F(7 ) and from 0.5786 to 0.9556 in wild population from Lake Liangzi. These markers are useful as tools for the detection of genetic variation levels in selected strains and wild populations of blunt snout bream for germplasm conservation.

Isolation and characterization of polymorphic microsatellite markers from Australia short-finned eel (Anguilla australis Richardson).

Seventeen microsatellite DNA loci from the Australian short-finned eel (Anguilla australis Richardson) were isolated and their amplification characteristics were described. The polymerase chain reaction primers were tested on 40 eel individuals. The primers amplified loci with relatively high numbers of alleles, ranging from five to 14 with an average of nine per locus. Mean observed heterozygosity (H(O) ) and expected heterozygosity (H(E) ) were 0.6779 and 0.7374, respectively, indicating that these markers would be useful for population studies. No loci deviated significantly from Hardy-Weinberg equilibrium (P = 0.05) and no evidence was found for genotypic disequilibrium among loci at a 5% significance level.

Hox gene clusters in blunt snout bream, Megalobrama amblycephala and comparison with those of zebrafish, fugu and medaka genomes.

Hox genes encode transcription factors that play a key role in specifying the body plan in metazoans and are therefore essential in explaining patterns of evolutionary diversity. While each Hox cluster contains the same genes among the different mammalian species, this does not happen in ray-finned fish, in which both the number and organization of Hox genes and even Hox clusters are variables. Here we reveal the organization of Hox genes loci in blunt snout bream. Forty-nine Hox genes including a pseudogene A9b in total have been found in seven clusters as follows: 8 Hox genes in the Aa cluster; 5 in Ab; 10 in Ba; 4 in Bb; 11 in Ca; 4 in Cb; and 7 in Da. In terms of gene content, clusters organization and sequence similarities of putative amino acids, blunt snout bream is more closely related to zebrafish than to fugu and medaka. In contrast to the situation in fugu and medaka, both blunt snout bream and zebrafish have duplicated HoxC cluster but only a single copy of the HoxD cluster. The result implies that the loss of the second HoxD cluster might be a shared feature of the Ostariophysi, to which zebrafish and blunt snout bream both belong. Phylogenetic analysis bases on the paralogous genes from twin clusters supports the duplication-first model, i.e., four original clusters may have duplicated in an event before the divergence of the blunt snout bream-plus-zebrafish lineage and the fugu-plus-medaka lineage. Additionally, the relationship between the decrease of GC level and the loss of conservation and function of one of the paralogous genes from twin clusters is discussed.

Structure of the mitochondrial DNA control region of the sinipercine fishes and their phylogenetic relationship.

The mitochondrial DNA control region of Siniperca chuatsi, S. kneri, S. scherzeri, S. obscura, S. undulata, Coreosiniperca roulei and Coreoperca whiteheadi were amplified by PCR amplification and directly sequenced. The mtDNA control region of the sinipercine fishes could be separated into three domains, namely, the terminal associated sequence domain, the central conserved sequence domain and the conserved sequence block domain. The extended terminal associated sequence (ETAS), three conserved sequence blocks (CSB-F, CSB-E, CSB-D) in the central conserved sequence domain and three conserved sequence blocks (CSB1, CSB2, CSB3) in the conserved sequence block domain were also identified. The phylogenetic relationships among these sinipercine fishes were constructed through neighbor-joining and maximum parsimony methods using Percidae and Serranidae as outgroups. Results showed that sinipercine fishes were a monophyletic group, with Siniperca forming one group, and Coreoperca forming another group. Coreosiniperca roulei did not form an independent group but was merged into the genus Siniperca. Thus it should be renamed as Siniperca roulei.

Genetic variability and relationships in mitochondrial DNA CO II gene sequence of red common carps in China.

Nucleotide sequence of Cytochrome coxidase subunit II (CO II) of mitochondrial DNA ( mtDNA) was analyzed from four populations of the red common carp (C. c. var. singuonensis, C. c. var. wananensis, C. c. var. wuyuanensis and C. c. var. color) as well as one population of wild common carp in China to detect their genetic variability and phylogenetic relationship. Nineteen nucleotide variable positions were detected in analyzed 607 bp length sequence and 15 haplotypes were defined among five populations,but only one shared haplotype was observed in C. c. var. singuonensis and C. c. var. color, respectively. The characteristic intrapopulation single nucleotide polymorphisms (SNP) were observed in 103th, 149th, 239th, 368th, 395th nucleotide position. The highest haplotypic diversity (h) and nucleotide diversity (pi) existed in C. c. var. wuyuanensis and the lowest exited in C. c. var. wananensis. The significant fixation indices (Fst) in most comparisons between populations were observed. The neighbor-joining trees from haplotypes and populations of red common carp indicated C. c. var singuonensis and C. c. var. color might have originated from one monophyletic group, while C. c. var. wananensis and C. c. var. wuyuanenesis might have originated from an independent evolutionary branch.